This research proposal will focus on the human MDR 1 gene as a model for maximizing gene expression in human hematopoietic cells following retroviral gene transfer. The MDR 1 gene may ultimately be used as a dominant marker to enrich for gene modified cells in vivo, or to protect bone marrow cells from the toxicity of chemotherapy. Both applications are dependent on achieving high level expression of MDR 1 in primitive stem cells and their committed progeny. Studies in animal models indicate that MDR retroviral vectors confer drug resistance to cells reconstituting hematopoiesis after transplant, but expression is attenuated by cryptic splicing within the vector. We have developed second generation vectors, designed to maximize expression in hematopoietic cells, and will characterize their expression in a human long-term bone marrow culture system. The specific aims of the proposal are: (1) to use site directed mutagenesis of the MDR 1 cDNA to maximize cellular drug resistance following retroviral gene transfer; (2) to develop a protocol for reproducible and efficient transduction of primitive human hematopoietic cells with retroviral vectors using bone marrow stromal support; (3) to develop a human long-term bone marrow culture model to examine expression of MDR retroviral vectors in hematopoietic progenitors; and (4) to determine if alternative MDR retroviral vectors based on Harvey murine sarcoma virus (HaMSV) and myeloproliferative sarcoma virus (MPSV) backbones will confer higher levels of drug resistance to human hematopoietic progenitors in long-term bone marrow culture. The information gained in these studies will also have more general value in designing retroviral vectors that direct high level gene expression in this specialized cell population.